Molecular cloning of a P-type Ca²⁺-ATPase from the halotolerant alga Dunaliella bioculata

Abstract

We have determined the complete primary structure of a putative P-type Ca²⁺ -ATPase from the unicellular, halotolerant alga Dunaliella bioculata. The protein (DBCA1) with a calculated molecular mass of 114 kDa and eight or ten putative transmembrane segments contains all amino acid motifs specific to the family of P-type ATPases. Highest homology scores were obtained by comparison with (sarco)endoplasmic reticulum-type plant and animal Ca²⁺-ATPases (54% identity, 70% similarity). In addition, all amino acids shown to be essential for Ca²⁺ transport in animal sarcoplasmic reticulum Ca²⁺-ATPases are preserved in DBCA1. Significantly lower homologies were found with animal plasma membrane Ca²⁺-ATPases (33% identity, 55% similarity), and the carboxyterminus of DBCA1 gave no indications of possible calmodulin-binding sites, characteristic of those enzymes. It is assumed that DBCA1 is a representative of the endomembrane class of Ca²⁺ ATPases. In Northern blot experiments with polyadenylated RNA, a 3.7-kb transcript was detected at levels which were very low compared with those of the plasma membrane H⁺-ATPase from D. bioculata (DBPMA1; Wolf et al., 1995, Plant Mol Biol 28: 657–666). Southern blot analyses suggest the existence of additional Ca²⁺-ATPase genes in the haploid genome of D. bioculata.